Organellar gene expression: Regulation of phage-type RNA polymerase transcript accumulation and analyses of mitochondrial gene copy numbers in Arabidopsis DISSERTATION zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Diplom-Biologe Tobias Preuten geboren am 29.05.1978 in Solingen Präsident der Humboldt-Universität zu Berlin Prof. Dr. Dr. h.c. Christoph Markschies Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. rer. nat., habil. Lutz-Helmut Schön Gutachter: 1. Prof. Dr. Thomas Börner 2. Prof. Dr. Axel Brennicke 3. Dr. Alisdair Fernie Tag der mündlichen Prüfung: 16.Oktober 2009 Let us imagine a palm tree, growing peacefully near a spring, and a lion, hiding in the brush nearby, all of its muscles taut, with bloodthirsty eyes, prepared to jump upon an antelope and to strangle it. The symbiotic theory, and it alone, lays bare the deepest mysteries of this scene, unravels and illuminates the fundamental principle that could bring forth two such utterly different entities as a palm tree and a lion. The palm behaves so peacefully, so passively, because it is a symbiosis, because it contains a plethora of little workers, green slaves (chromatophores) that work for it and nourish it. The lion must nourish itself. Let us imagine each cell of the lion filled with chromatophores, and I have no doubt that it would immediately lie down peacefully next to the palm, feeling full, or needing at most some water with mineral salts. Mereschkowsky, C. (1905). Über Natur und Ursprung der Chromatophoren im Pflanzenreiche. Biol. Centralbl., 25: 593–604. English translation in Martin, W., Kowallik, K. V. (1999). Annotated English translation of Mereschkowsky's 1905 paper ‘Über Natur und Ursprung der Chromatophoren im Pflanzenreiche’. Eur. J. Phycol., 34: 287–295. ABSTRACT Abstract In addition to eubacterial-like multi-subunit RNA polymerases (RNAP) localized in plastids and the nucleus, Arabidopsis thaliana contains three phage-like single-unit, nuclear- encoded, organellar RNAPs. The enzymes RpoTp and RpoTm are imported into plastids and mitochondria, respectively, whereas RpoTmp shows dual targeting properties into both organelles. To investigate if expression of the RpoT genes is light-dependent, light-induced transcript accumulation of RpoTm, RpoTp and RpoTmp was analyzed using quantitative real-time-PCR in 7-day-old seedlings as well as in 3- and 9-week-old rosette leaves. To address the question whether RpoT transcript accumulation is regulated differentially during plant development transcript abundance was measured during leaf development. Additionally, effects of the plants circadian rhythm on RpoT transcript accumulation were analyzed. Transcripts of all three RpoT genes were found to be strongly light-induced even in senescent leaves and only marginally influenced by the circadian clock. Further analyses employing different photoreceptor mutants and light qualities revealed the involvement of multiple receptors in the light-induction process. The biogenesis of mitochondria and chloroplasts as well as processes like respiration and photosynthesis require the activity of genes residing in at least two distinct genomes. There have to be ways of intracellular communication between different genomes to control gene activities in response to developmental and metabolic needs of the plant. In this study, it was shown that gene copy numbers drastically increased in photosynthetically inactive Arabidopsis seedlings. Mitochondrial DNA contents in cotyledons and leaves ranging in age from 2-day-old cotyledons to 37-day-old senescent rosette leaves were examined. A common increase in senescing rosette leaves and drastic differences between individual genes were found, revealing the importance of an integrative chondriome in higher plant cells. Keywords: phage-type RNA polymerase, light-induction, photoreceptors, mitochondrial gene copy numbers, chondriome ABSTRACT Abstract Zusätzlich zu der eubakteriellen RNA-Polymerase (RNAP) der Plastiden sind im Zellkern von Arabidopsis thaliana drei weitere, phagentypische RNAP kodiert, die jeweils aus nur einer Einheit aufgebaut sind. Die Enzyme RpoTp und RpoTm werden in die Plastiden, bzw. die Mitochondrien transportiert, während RpoTmp in beiden Organellen zu finden ist. Um die Lichtabhängigkeit der RpoT-Gene zu untersuchen, wurde die lichtinduzierte Akkumulation ihrer Transkripte in 7-Tage alten Keimlingen, sowie 3- bzw. 9-Wochen alten Rosettenblättern mittels quantitativer real-time PCR ermittelt. Die entwicklungsabhängige Regulation der RpoT-Transkript-Akkumulation wurde außerdem während der Blattentwicklung analysiert. Zusätzlich wurde der Einfluss des circadianen Rhythmus untersucht. Es stellte sich heraus, dass die Transkriptakkumulation aller drei RpoT-Gene stark lichtinduziert war und nur marginalen circadianen Schwankungen unterlag. In weiteren Versuchen mit verschiedenen Lichtrezeptor-Mutanten und unterschiedlichen Lichtqualitäten wurde der Einfluss multipler Rezeptoren auf den Prozess der Lichtinduktion gezeigt. In den Zellen höherer Pflanzen finden sich drei Genome. Die Biogenese von Chloroplasten und Mitochondrien, sowie lebenswichtige Prozesse, wie Atmung und Photosynthese setzen oftmals die Aktivität von Genen auf mindestens zwei dieser Genome voraus. Eine intrazelluläre Kommunikation zwischen den verschiedenen Genomen ist daher unumgänglich für einen funktionierenden Stoffwechsel der Pflanze. In dieser Arbeit wurde herausgestellt, dass die Zahl mitochondrialer Genkopien in photosynthetisch inaktiven Arabidopsis-Keimlingen drastisch erhöht ist. Bei der Untersuchung des DNA-Gehaltes in Proben, die Altersstufen von 2-Tage alten Keimblättern bis hin zu 37-Tage alten, seneszenten Rosettenblättern umfassten, fand sich ein deutlicher Anstieg der Kopienzahlen in älteren Rosettenblättern. Außerdem unterschieden sich die Kopienzahlen der untersuchten Gene zum Teil erheblich voneinander. Schlagworte: Phagentyp-RNA-Polymerasen, Lichtinduktion, Photorezeptoren, mitochondriale Genkopienzahlen, Chondriom TABLE OF CONTENTS Table of contents Zusammenfassung..........................................................................................................................................I Summary ....................................................................................................................................................... II 1. Introduction .......................................................................................................................................... 1 1.1 The origin of organelles and their roles in higher plants……………………………………………1 1.1.1 Mitochondria ............................................................................................................................ 1 1.1.2 Plastids ..................................................................................................................................... 4 1.2 Higher plant organellar genomes…………………………………………………………………... 8 1.2.1 The chondrome of higher plants .............................................................................................. 8 1.2.2 The plastome .......................................................................................................................... 10 1.3 The plant chondriome 12 1.4 Organellar transcription and phage-type RNA polymerases……………………………………... 14 1.4.1 The transcription machinery of plant mitochondria............................................................... 14 1.4.2 The transcription machinery of plastids................................................................................. 17 1.4.3 Regulation of organellar gene expression by phage-type RNA polymerases........................ 19 1.5 Aim of this work…………………………………………………………………………………. 22 2 Materials and Methods ...................................................................................................................... 24 2.1 Materials 24 2.1.1 Providers ................................................................................................................................ 24 2.1.2 Plant material ......................................................................................................................... 25 2.1.3 Bacterial strains...................................................................................................................... 25 2.1.4 Oligonucleotides .................................................................................................................... 25 2.1.5 Software ................................................................................................................................. 25 2.2 Methods…………………………………………………………………………………………... 26 2.2.1 Plant growth ........................................................................................................................... 26 2.2.2 Surface sterilization of Arabidopsis seeds ............................................................................. 27 2.2.3 Isolation of nucleic acids........................................................................................................ 27 2.2.3.1 Isolation of genomic DNA ........................................................................................... 27 2.2.3.2 Isolation of total RNA .................................................................................................. 27 2.2.4 Gel electrophoresis